In scanning microscopy, a specimen is illuminated with a light beam in order to observe the reflected or fluorescent light emitted from the specimen. The focus of the illumination light beam is moved in an object plane with the aid of a controllable beam deflection device, generally by tilting two mirrors, the deflection axes usually being perpendicular to one another so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the light coming from the specimen is measured as a function of the position of the scanning beam.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam. A confocal scanning microscope generally encompasses a light source, a focusing optical system with which the light of the source is focused onto an aperture (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detected or fluorescent light. The illumination light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen travels back via the beam deflection device to the beam splitter, traverses it, and is then focused onto the detection pinhole behind which the detectors are located. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a single-point datum is obtained that results, by sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers.
The samples to be examined are generally equipped with a marking, usually a fluorescent dye, that is optically excitable. These dyes can also be, for example, GFP (green fluorescence protein) or CFP (cyan fluorescence protein).
For many experiments on and examinations of biological samples, it is necessary, in addition to the microscopic observation of the sample, also to perform a manipulation of the sample. U.S. application Ser. No. 2002 0196535 A1 discloses a confocal scanning microscope with which at least one region of a sample (region of interest, ROI) can be both manipulated and observed. The manipulation, and the scanning of the region necessary for observation, are accomplished sequentially; for example, manipulation can occur in the forward direction in a line, while in the return direction the region of interest, and if applicable the surrounding area, are scanned for observation.
DE 100 43 986 A1 discloses a method for examining a sample by means of a confocal scanning microscope, in which firstly a preview image of the sample is acquired and then one or more regions of interest can be marked. Each region has specific illumination light beam wavelengths and/or illumination light beam power levels allocated to it, so that the sample can then be manipulated in the marked regions in accordance with the allocation.
For some experiments it is desirable to be able simultaneously to observe and manipulate the sample. A laser scanning microscope that permits simultaneous scanning and manipulation of a sample is known from U.S. Pat. No. 6,094,300. The laser scanning microscope contains two mutually independent beam deflection devices: one of the beam deflection devices guides the manipulation light beam over or through the sample, while the other beam deflection device directs the observation illumination light beam over or through the sample. The laser scanning microscope has the disadvantage that the light beams, namely the manipulation light beam and the scanning illumination light beam, coming from the beam deflection devices must be combined into one shared beam path, using a beam combiner, before entry into the objective. A very particular disadvantage of the necessary beam combiner is that the interference bands produced thereby in the image change during the scanning procedure because of the changes in the angle of incidence of the moving beam, and thus cannot be compensated for or calculated out in the context of image processing. The beam combiner furthermore produces a beam offset and thus results in considerable light losses.
DE 100 39 520 A1 likewise makes known a confocal scanning microscope with the capability for simultaneous manipulation and object detection. Two beam deflection devices are provided in this scanning microscope as well, one for the manipulation light beam and one for the illumination light beam. In a particular variant embodiment of this scanning microscope, the manipulation light beam is coupled into the beam path of the illumination light beam by means of the deflection mirror associated with the illumination light beam. The deflection mirror is embodied to be transparent to light having the wavelength of the manipulation light beam, and reflective for light having the wavelength of the illumination light beam.